In higher eukaryotes, secreted proteins including peptide hormones and neurotransmitters are synthesized as larger precursors from which they are released in mature form by the action of specific processing proteases (Steiner et al., Ann. N.Y. Acad Sci. 343 1-16, 1980; Douglass et al., Ann Rev. Biochem. 53, 665-715, 1984; Lynch and Snyder, Ann Rev. Biochem. 55 773-799, 1986). Many of these biologically active peptides are flanked within a precursor molecule by pairs of basic amino-acids; endoproteolytic cleavage after these residues is thought to be the initial step in precursor maturation. Further processing often requires removal of the basic amino-acids by a carboxypeptidase B-like enzyme. A metalloprotease showing specificity towards C-terminal arginine or lysine residues was purified from bovine adrenal chromaffin granules (Fricker and Snyder, J. Biol. Chem. 258, 10950-10955, 1983) and recently the gene encoding this protease (named carboxypeptidase E or enkephalin convertase) has been cloned (Fricker et al., Nature 323 461-464, 1986). This enzyme is believed to be involved in a similar processing step, to the KEX1 gene product, in mammalian cells. U.S. Pat. No. 3,625,829 teaches the purification of carboxypeptidase B from pancreas extracts.
The analogous proteolytic maturation of precursor molecules also occurs in the yeast Saccharomyces cerevisiae. The .alpha.-factor pheromone and K1 killer toxin are the best characterized examples of secreted proteins derived from such processed precursors. Yeast .alpha.-factor is a peptide pheromone of 13 amino-acid residues involved in the mating response of haploid cells. It is encoded by two genes: MF .alpha. 1 and MF .alpha. 2 (Kurjan and Herskowitz, Cell 30, 933-943, 1982; Singh et al., Nucleic Acids Res. 11, 4049-4063, 1983). The .alpha.-factor coding sequence occurs repetitively, with either four or two copies within the respective 165 or 120 amino acid precursors encoded by these genes. Biologically active pheromone peptides are separated in the precursors by LysArgGluAla(Glu/Asp)AlaGluAla spacers. The liberation of the pheromone requires the activity of three proteolytic enzymes: an endoprotease recognizing a pair of basic amino-acids, a dipeptidyl aminopeptidase capable of removing N-terminal GluAla repeats and a carboxypeptidase responsible for cleaving C-terminal basic residues (See FIG. 1). The first two enzymes have been identified as the KEX2 and the STE13 gene products, respectively, and both genes have been cloned (Julius et al, Cell 36 309-318, 1983, Cell 37 1075-1089, 1984). A possible candidate for the carboxypeptidase has been suggested on the basis of biochemical evidence (Achstetter and Wolf, EMBO J. 4 173-177, 1985).
The set of processing proteases required for secretion of killer toxin (a small protein secreted by yeast carrying linear M1 double stranded RNA) is less well characterized. The killer toxin protein has been cloned (Thomas et al., U.S. application Ser. No. 06/600,964, filed 16 Apr. 1984). Mature killer toxin consists of two subunits: .alpha. and .beta., which are separated within a precursor by a glycosylated .gamma. region (Bostian et al., Cell 36, 741-751, 1984; Skipper et al., EMBO J. 3 107-111, 1984; and see FIG. 1). Additionally the .alpha. subunit is preceded by a 44 amino-acid residue leader, the first 26 residues of which are a signal peptide removed during entry into the endoplasmic reticulum (Lolle and Bussey, Mol. Cell. Biol. 6 4274-4280, 1986). In addition to the signal peptidase, at least three other proteolytic events are necessary to release mature toxin from the precursor: (1) cleavage between the leader remnant and the .alpha. subunit (at the P2 site) to generate the authentic N-terminus of the .alpha.-subunit, (2) cleavage between the .alpha. and .gamma. peptides, (3) cleavage between the .gamma. and .beta. subunits (See FIG. 1). The last cleavage (3) occurs after LysArg residues of the .gamma. peptide and is probably carried out by the KEX2 coded protease. The fact that killer toxin is not secreted from kex2 mutants harboring the M1 dsRNA (Leibowitz and Wickner, Proc. Natl. Acad. Sci. U.S.A. 73 2061-2065, 1976; Bussey et al., Mol. Cell. Biol. 3 1362-1370, 1983) is consistent with this scheme. Enzymes responsible for events (1) and (2) have not been identified.
In the present invention we have focused on the KEX1 gene product. Mutations in this gene lead to failure to process the protoxin (Wickner and Leibowitz, Genetics 82 429-442, 1976; Bussey et al op. cit., 1983) and like kex2 mutations result in a killer minus phenotype. We expected that the KEX1 gene would encode a protease involved in releasing the subunit from the precursor molecule. An endoproteolytic, chymotrypsin-like activity was proposed for this protein based on inhibitor studies (Bussey et al. op. cit., 1983) and a cleavage site in the protoxin was postulated (Bostian et al. op. cit., 1984). To explore this possibility, we decided to clone the KEX1 gene and to characterize the encoded protein. Nucleotide sequence revealed a surprising feature of the gene product, namely extensive homology with carboxypeptidase Y including homology with residues at the active site. We have also found that kex1.sup.- null mutations are pleiotropic, affecting not only maturation of the killer toxin, but also of active .alpha.-factor mating pheromone. We have found that the KEX1 gene product acts as a carboxypeptidase B-like processing protease in the maturation of these precursors in yeast.
Carboxypeptidase Y (CPY) is a well characterized yeast vacuolar serine protease (Hayasi et al. J. Biol. Chem. 250, 5221-5226, 1975; Martin et-al., Carlsberg Res. Commun. 47, 1-3, 1982; Svendsen et al. Carlsberg. Res. Commun. 47, 15-27, 1982; and see Breddam, Carlsberg Res. Commun. 5, 83-128, 1985, for a review). This yeast protease is only known to be involved as a degradative enzyme and is not involved in protein processing. The cellular location of carboxypeptidase Y and the KEX1 product is different, CPY being found in yeast vacuoles and the KEX1 gene product is thought to be located in the Golgi apparatus. These two protease are also genetically unrelated; CPY cannot complement kex1 mutations.
KEX1 and KEX2 are required for the processing of some proteins and peptides of commercial importance, for example hormones and neuropeptides. Therefore, an application of this present invention is to provide these processing proteases in combination with commercially important proteins and peptides, either together in the same vector or on separate vectors. These processing proteases would provide for the specific processing of the desired secretion polypeptides to yield mature proteins.